It has been well-known that nucleic acid molecules such as DNA and RNA have a function of producing proteins through transcription/translation. However, a large number of molecular species (such as ribozymes and RNAi) having a function due to interaction with proteins and high-molecular-weight substances (i.e. a function that is different from the originally observed function of nucleic acid molecules) have been discovered, and their application to the therapeutic field, etc. attracts attention. In particular, aptamers attract attention as molecular species which exhibit their function by binding directly to target substances such as proteins, high-molecular-weight materials, pharmaceuticals, etc. At present, a SELEX (Systematic Evolution of Ligands by EXponential enrichment) method is known as a general method of obtaining aptamers (see Patent Document 1). The aptamers and the SELEX method are described in detail in Non-Patent document 1.
One feature of aptamers obtained by the SELEX method is that aptamers are composed of an arbitrary primer sequence and a random sequence of arbitrary length. Another feature of aptamers obtained by SELEX method is that, even if promising candidates of aptamers are screened sufficient number of times in the SELEX method, there are plural types of finally-obtained aptamers.
It has been known that aptamers having such features includes many redundant regions such as a primer sequence and that the region necessary for the aptamer to actually bind to the target substance is a part of bases constituting the aptamer. Therefore, identification of the region necessary for the aptamer to bind to the target substance is very important not only in improving the efficiency of production of aptamers but also in understanding the mode of binding of the aptamer to the target substance.
However, in order to identify the region necessary for the aptamer to bind to the target substance, it has been conventionally required to conduct a binding experiment in vitro or the like using an obtained aptamer and its target substance. For example, redundant sequences that are considered unnecessary for the obtained aptamers to bind to their target substances are removed from the aptamers by way of digestion according to a genetic engineering technique so as to prepare a series of nucleotide sequences, and it is required to conduct a binding experiment or the like using the nucleotide sequences and the target substances in order to identify region necessary for binding. Such a method involves not only a large amount of labor, and after all requires trial and error based on experimenter's experience (for example, how much obtained aptamers need to be digested). Therefore, a method for efficiently identifying a region of the aptamer which is essential for binding to the target substance has been sought.
Patent Document 1: Published Japanese Translation No. H05-507413 of the PCT International Publication.
Non-Patent Document 1: “Biotechnology Series: Frontier of RNA Engineering” edited by Yoshikazu Nakamura and Shoji Ochi, CMC Publishing Co., Ltd, pp. 139-141.